The male brain contains significant concentrations of 17b-estradiol (E2), due to conversion from testosterone by aromatase, and estrogen receptors that upon activation initiate a number of signaling pathways implicated in the treatment of depression. However, the effects of E2 on male behavior in animal models of depression and anxiety have received only limited consideration. Similarly, the potential beneficial effects of E2 in male humans have been studied only to a limited degree, in part due to its potential side effects including feminization. As such, brain-selective E2 therapy could, for some patients, be an improvement over existing treatments, which result in inadequate treatment for the majority of patients. Therefore, there is a huge unmet need for a new and safer E2 therapy for preclinical studies, as well as human male proof of concept and future clinical treatment studies. Herein, we propose to test, in animal models of depression and anxiety, 10b,17b- dihydroxyestra-1,4-dien-3-one (DHED)-an innovative compound classified according to its unique mechanism of action as a brain-selective bioprecursor prodrug of E2. Based on encouraging preliminary data, we aim at establishing the possible benefits of brain-selective E2 in the treatment of depression and anxiety in males. Our central hypothesis is that brain selective E2 treatment wil result in antidepressant- and anxiolytic-like effects in male mice. This hypothesis is supported by our studies where administration of DHED to male and female rodents produced significant E2 levels in the brain. Further, our preliminary data indicates that administration of DHED has antidepressant- and anxiolytic-like effects in female mice and is likely to have similar effects in male mice. In the first Specific Aim, we will define the range of brain-selective E2 treatment with DHED on depression- and anxiety-like behaviors in male mice. These studies will encompass acute and chronic administrations, dose-response curves, and studies involving gonadally intact and orchidectomized males. We hypothesize that E2 formed from the systemically administered DHED will result in robust antidepressant- and anxiolytic-like effects in male mice and that E2 will reverse deleterious effects of orchidectomy on depression and anxiety outcomes. In the second Specific Aim, we will identify, through bioanalytical methods, brain-selective DHED distribution and bioactivation to E2 that associate with behaviors and intracellular signaling pathway activity. We will focus on the measurement of E2 in target (CNS) and non- target (peripheral) tissues and in the circulation after acute and chronic systemic administration. We wil correlate all behavioral outcomes in Specific Aim 1 with brain levels of E2, which will be the first study ever in males to link brain concentration of E2 to anxiety- and depression-related behavioral outcomes. Successful completion of the proposed experiments will allow us to conclude whether to move forward with brain-selective E2 therapy as a potential treatment of depression and anxiety in men, and provide proof-of-concept to support future rodent and human testing of brain selective E2 in various other CNS diseases in males.